Alterations in gene expression and activity during squamous cell carcinoma development

This study focuses on characterizing the genetic and biological alterations associated with squamous cell carcinoma development. Normal human epidermal keratinocytes (HEKs), cells isolated from a preneoplastic lesion (IEC-1), and two neoplastic cell lines, SCC-25 and COLD-16, were grown as raft cultures, and their gene expression profiles were screened using cDNA arrays. Our data indicated that the expression levels of at least 37 genes were significantly (P less than or equal to 0.05; 1.9% of genes screened) altered in neoplastic cells compared with normal cells. Of these genes, 10 genes were up-regulated and 27 genes were down-regulated in the neoplastic cells. In addition, 51% of the genes altered in the neoplastic cells were already altered in the preneoplastic IEC-1 cells. Immunohistochemical staining of patient tumors was used to verify the cDNA array analysis. Our analysis indicated that alterations in genes associated with extracellular matrix production and apoptosis are disrupted in preneoplastic cells, whereas later stages of neoplasia are associated with alterations in gene expression for genes involved in DNA repair or epidermal growth factor (EGF) receptor/mitogen-activated protein kinase kinase (MAPKK)/MAPK/activator protein-1 (AP-1) signaling. Subsequent functional analysis of the alterations in expression of the EGF receptor/MAPKK/MAPK/AP-1 genes suggested they did not contribute to the neoplastic phenotype.

Differences in mouse strain influence leukocyte and immunoglobulin phenotype response to Porphyromonas gingivalis

This study examined the nature of the infiltrating cells in Porphyromonas gingivalis-induced lesions and immunoglobulins in the serum samples of BALB/c (H-2(d)), C57BL6 (H-2(b)), DBA/2J (H-2(d)) and CBA/CaH (H-2(k)) mice. Mice were immunized intraperitoneally with P. gingivalis outer membrane antigens or sham-immunized with phosphate-buffered saline followed by subcutaneous challenge with live organisms 1 week after the final immunization. The resulting skin abscesses were excised 7 days later, cryostat sections cut and an immunoperoxidase method used to detect the presence of CD4(+) and CD8(+) T-cell subsets, CD14(+) macrophages and CD19(+) B cells. Peroxidase positive neutrophils and IgG1- and IgG2a-producing plasma cells were also identified. Anti P. gingivalis IgG1 and IgG2a subclass antibodies were determined in serum obtained by cardiac puncture. Very few CD8(+) T cells and CD19(+) B cells were found in any of the lesions. The percentages of CD4(+) cells, CD14(+) cells and neutrophils were similar in lesions of immunized BALB/c and C57BL6 mice, with a trend towards a higher percentage of CD14(+) cells in sham-immunized mice. The percentage of CD14(+) cells was higher than that of CD4(+) cells in immunized compared with sham-immunized DBA/2J mice. The percentages of CD4(+) and CD14(+) cells predominated in immunized CBA/CaH mice and CD4(+) cells in sham-immunized CBA/CaH mice. The percentage of neutrophils in immunized CBA/CaH mice was significantly lower than that of CD14(+) cells and CD4(+) cells in sham-immunized mice. IgG1(+) plasma cells were more dominant than IgG2a(+) cells in immunized BALB/c, C57BL6 and DBA/2J mice, whereas IgG2a(+) plasma cells were more obvious in sham-immunized mice. IgG2a(+) plasma cells were predominant in immunized and sham-immunized CBA/CaH mice. In the serum, specific anti-P. gingivalis IgG2a antibody levels (Th1 response) were higher than IgG1 levels (Th2 response) in sham-immunized CBA/CaH and DBA/2J mice. In immunized BALB/c mice, IgG2a levels were lower than IgG1 levels, while IgG2a levels were higher in immunized C57BL6 mice. In conclusion, this study has shown differences in the proportion of infiltrating leukocytes and in the subclasses of immunoglobulin produced locally and systemically in response to P. gingivalis in different strains of mice, suggesting a degree of genetic control over the response to P. gingivalis.

Proteasomal targeting of a viral oncogene abrogates oncogenic phenotype and enhances immunogenicity

The ability of viral or mutated cellular oncogenes to initiate neoplastic events and their poor immunogenicity have considerably undermined their potential use as immunotherapeutic tools for the treatment of human cancers. Using an EpsteinBarr virus-encoded oncogene, latent membrane protein 1 (LMP1), as a model, we report a novel strategy that both deactivates cellular signaling pathways associated with the oncogenic phenotype and reverses poor immunogenicity. We show that cotranslational ubiquitination combined with Wend rule targeting of LMP1 enhanced the intracellular degradation of LMP1 and total blockade of LMP1-mediated nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription (STAT) activation in human cells. In addition, although murine cells expressing LMP1 were uniformly tumorigenic, this oncogenicity was completely abrogated by covalent linkage of LMP1 with ubiquitin, while an enhanced CD8(+) T cell response to a model epitope fused to the C-terminus of LMP1 was observed following immunization with ubiquitinated LMP1. These observations suggest that proteasomal targeting of tumor-associated oncogenes could be exploited therapeutically by either gene therapy or vaccination.

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4(+) T cells, have never been defined. We generated CD4+ T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-gamma, and tumor necrosis factor-a, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.

Expression of the CD44v2-10 isoform confers a metastatic phenotype: Importance of the heparan sulfate attachment site CD44v3

We expressed the full-length CD44v2-10 isoform in SKHep1 cells, a nonmetastatic human hepatocellular carcinoma cell line that does not express any endogenous CD44v isoforms. In SCID mice, expression of CD44v2-10 by SKHep1 cells had no effect on s.c. primary tumor development but caused pulmonary metastases in 41% (7 of 17) of animals compared with control SKHep1 cells (0 of 16; P < 0.01). CD44v2-10 expression by SKHep1 cells resulted in enhanced heparan sulfate (HS) attachment and an enhanced capacity to bind heparin-binding growth factors. Mutation of the v3 domain to prevent HS attachment and growth factor binding abolished the metastatic phenotype, demonstrating that HS modification of CD44v2-10 plays a critical role in the development of metastases in this model. However, in vitro proliferation, motility, and invasion were not altered by CD44v2-10 expression.

Antiproliferative and phenotype-transforming antitumor agents derived from cysteine

Selective destruction of malignant tumor cells without damaging normal cells is an important goal for cancer chemotherapy in the 21st century. Differentiating agents that transform cancer cells to either a nonproliferating or normal phenotype could potentially be tissue-specific and avoid side effects of current drugs. However, most compounds that are presently known to differentiate cancer cells are histone deacetylase inhibitors that are of low potency or suffer from low bioavailability, rapid metabolism, reversible differentiation, and nonselectivity for cancer cells over normal cells. Here we describe 36 nonpeptidic compounds derived from a simple cysteine scaffold, fused at the C-terminus to benzylamine, at the N-terminus to a small library of carboxylic acids, and at the S-terminus to 4-butanoyl hydroxamate. Six compounds were cytotoxic at nanomolar concentrations against a particularly aggressive human melanoma cell line (MM96L), four compounds showed selectivities of greater than or equal to5:1 for human melanoma over normal human cells (NFF), and four of the most potent compounds were further tested and found to be cytotoxic for six other human cancer cell lines (melanomas SK-MEL-28, DO4; prostate DU145; breast MCF-7; ovarian JAM, CI80-13S). The most active compounds typically caused hyperacetylation of histones, induced p21 expression, and reverted phenotype of surviving tumor cells to a normal morphology. Only one compound was given orally at 5 mg/kg to healthy rats to look for bioavailaiblity, and it showed reasonably high levels in plasma (C-max 6 mug/mL, T-max 15 min) for at least 4 h. Results are sufficiently promising to support further work on refining this and related classes of compounds to an orally active, more tumor-selective, antitumor drug.

Disruption of the BLM gene in ATM-null DT40 cells does not exacerbate either phenotype

Bloom syndrome and ataxia-telangiectasia are autosomal recessive human disorders characterized by immunodeficiency, genome instability and predisposition to develop cancer. Recent data reveal that the products of these two genes, BLM and ATM, interact and function together in recognizing abnormal DNA structures. To investigate the function of these two molecules in DNA damage recognition, we generated double knockouts of ATM(-/-) BLM-/- in the DT40 chicken B-lymphocyte cell line. The double mutant cells were viable and exhibited a variety of characteristics of both ATM(-/-) and BLM-/- cells. There was no evidence for exacerbation of either phenotype; however, the more extreme radiosensitivity seen in ATM(-/-) and the elevated sister chromatid exchange seen in BLM-/- cells were retained in the double mutants. These results suggest that ATM and BLM have largely distinct roles in recognizing different forms of damage in DNA, but are also compatible with partially overlapping functions in recognizing breaks in radiation-damaged DNA.

Herpesvirus-specific CD8 T cell immunity in old age: Cytomegalovirus impairs the response to a coresident EBV infection

Aging in humans is associated with increased infections and the reduced proliferative capacity of T cells, part of the more global phenomenon termed immune senescence. The etiology of immune senescence is unknown but the accumulation of virus-specific memory T cells may be a contributory factor. We have examined CD8 T cell responses to two persistent herpesvirus infections, CMV and EBV, and to a recurrent virus infection, influenza, in different age cohorts of healthy donors using HLA-peptide tetramers and intracellular cytokine detection. Of these, CMV appears to be the most immunogenic, with the CD8 T cell response representing over 10% of the CD8 pool in many elderly donors. Interestingly, the effect of age upon EBV-specific responses depends upon donor CMV sero-status. In CMV seropositive donors, the magnitude of the EBV-specific immune response is stable with age, but in CMV seronegative donors, the response to EBV increases significantly with age. By contrast, the influenza-specific CD8 T cell immune response decreases with age, independent of CMV status. The functional activity of the herpesvirus-specific immune response decreases in elderly donors, although the characteristic phenotypes of CMV- and EBV-specific memory populations are retained. This demonstrates that aging is associated with a marked accumulation of CMV-specific CD8 T cells together with a decrease in immediate effector function. Moreover, infection with CMV can reduce prevailing levels of immunity to EBV, another persistent virus. These results suggest that carriage of CMV may be detrimental to the immunocompetent host by suppressing heterologous virus-specific immunity during aging.

Identifying the molecular phenotype of renal progenitor cells

Although many of the molecular interactions in kidney development are now well understood, the molecules involved in the specification of the metanephric mesenchyme from surrounding intermediate mesoderm and, hence, the formation of the renal progenitor population are poorly characterized. In this study, cDNA microarrays were used to identify genes enriched in the murine embryonic day 10.5 (E10.5) uninduced metanephric mesenchyme, the renal progenitor population, in comparison with more rostral derivatives of the intermediate mesoderm. Microarray data were analyzed using R statistical software to determine accurately genes differentially expressed between these populations. Microarray outliers were biologically verified, and the spatial expression pattern of these genes at E10.5 and subsequent stages of early kidney development was determined by RNA in situ hybridization. This approach identified 21 genes preferentially expressed by the E10.5 metanephric mesenchyme, including Ewing sarcoma homolog, 14-3-3 theta, retinoic acid receptor-alpha, stearoyl-CoA desaturase 2, CD24, and cadherin-11, that may be important in formation of renal progenitor cells. Cell surface proteins such as CD24 and cadherin-11 that were strongly and specifically expressed in the uninduced metanephric mesenchyme and mark the renal progenitor population may prove useful in the purification of renal progenitor cells by FACS. These findings may assist in the isolation and characterization of potential renal stem cells for use in cellular therapies for kidney disease.

An in vivo comparative study of sonic, desert and Indian hedgehog reveals that hedgehog pathway activity regulates epidermal stem cell homeostasis

Despite the well-characterised role of sonic hedgehog (Shh) in promoting interfollicular basal cell proliferation and hair follicle downgrowth, the role of hedgehog signalling during epidermal stem cell fate remains largely uncharacterised. In order to determine whether the three vertebrate hedgehog molecules play a role in regulating epidermal renewal we overexpressed sonic (Shh), desert (Dhh) and Indian (Ihh) hedgehog in the basal cells of mouse skin under the control of the human keratin 14 promoter. We observed no overt epidermal morphogenesis phenotype in response to Ihh overexpression, however Dhh overexpression resulted in a range of embryonic and adult skin manifestations indistinguishable from Shh overexpression. Two distinct novel phenotypes were observed amongst Shh and Dhh transgenics, one exhibiting epidermal progenitor cell hyperplasia with the other displaying a complete loss of epidermal tissue renewal indicating deregulation of stem cell activity. These data suggest that correct temporal regulation of hedgehog activity is a key factor in ensuring epidermal stem cell maintenance. In addition, we observed Shh and Dhh transgenic skin from both phenotypes developed lesions reminiscent of human basal cell carcinoma (BCC), indicating that BCCs can be generated despite the loss of much of the proliferative (basal) compartment. These data suggest the intriguing possibility that BCC can arise outside the stem cell population. Thus the elucidation of Shh (and Dhh) target gene activation in the skin will likely identify those genes responsible for increasing the proliferative potential of epidermal basal cells and the mechanisms involved in regulating epidermal stem cell fate.

Contemporaneous fluctuations in T cell responses to persistent herpes virus infections

The classical paradigm for T cell dynamics suggests that the resolution of a primary acute virus infection is followed by the generation of a long-lived pool of memory T cells that is thought to be highly stable. Very limited alteration in this repertoire is expected until the immune system is re-challenged by reactivation of latent viruses or by cross-reactive pathogens. Contradicting this view, we show here that the T cell repertoire specific for two different latent herpes viruses in the peripheral blood displayed significant contemporaneous co-fluctuations of virus-specific CD8(+) T cells. The coordinated responses to two different viruses suggest that the fluctuations within the T cell repertoire may be driven by sub-clinical viral reactivation or a more generalized 'bystander' effect. The later contention was supported by the observation that, while absolute number of CD3(+) T cells and their subsets and also the cell surface phenotype of antigen-specific T cells remained relatively constant, a loss of CD62L expression in the total CD8(+) T cell population was coincident with the expansion of tetramer-positive virus-specific T cells. This study demonstrates that the dynamic process of T cell expansion and contractions in persistent viral infections is not limited to the acute phase of infection, but also continues during the latent phase of infection.

Typical medullary breast carcinomas have a basal/myoepithelial phenotype

Medullary breast cancer (MBC) is a rare, diagnostically difficult, pathological subtype. Despite being high grade, it has a good prognosis. MBC patients have an excess of BRCA1 germ-fine mutation and reliable identification of MBC could help to identify patients at risk of carrying germline BRCA1 mutations or in whom chemotherapy could be avoided. The aim of this study was therefore to improve diagnosis by establishing an MBC protein expression profile using immunohistochemistry (IHC) on tissue-microarrays (TMA). Using a series of 779 breast carcinomas ('EC' set), diagnosed initially as MBC, a double-reading session was carried out by several pathologists on all of the histological material to establish the diagnosis as firmly as possible using a 'medullary score'. Only MBCs with high scores, i.e. typical MBC (TMBC) (n = 44) and non-TMBC grade III with no or low scores (n = 160), were included in the IHC study. To validate the results obtained on this first set, a control series of TMBC (n = 17) and non-MBC grade III cases (n = 140) ('IPC' set) was studied. The expression of 18 proteins was studied in the 61 TMBCs and 300 grade III cases from the two sets. The global intra-observer concordance of the first reading for the diagnosis of TMBC was 94%, with almost perfect kappa (kappa) of 0.815. TMBC was characterized by a high degree of basal/myoepithelial differentiation. In multivariate analysis with logistic regression, TMBC was defined by the association of P-cadherin (R = 2.29), MIB1 > 50 (R = 3.80), ERBB2 negativity (R = 2.24) and p53 positivity (RR = 1.45). Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cosuppression of eukaryotic release factor 1-1 in Arabidopsis affects cell elongation and radial cell division

The role of the eukaryotic release factor 1 (eRF1) in translation termination has previously been established in yeast; however, only limited characterization has been performed on any plant homologs. Here, we demonstrate that cosuppression of eRF1-1 in Arabidopsis (Arabidopsis thaliana) has a profound effect on plant morphology, resulting in what we term the broomhead phenotype. These plants primarily exhibit a reduction in internode elongation causing the formation of a broomhead-like cluster of malformed siliques at the top of the inflorescence stem. Histological analysis of broomhead stems revealed that cells are reduced in height and display ectopic lignification of the phloem cap cells, some phloem sieve cells, and regions of the fascicular cambium, as well as enhanced lignification of the interfascicular fibers. We also show that cell division in the fascicular cambial regions is altered, with the majority of vascular bundles containing cambial cells that are disorganized and possess enlarged nuclei. This is the first attempt at functional characterization of a release factor in vivo in plants and demonstrates the importance of eRF1-1 function in Arabidopsis.

Specialization in pyramidal cell structure in the sensory-motor cortex of the chacma baboon (Papio ursinus) with comparative notes on macaque and vervet monkeys

The systematic study of pyramidal cell structure has revealed new insights into specialization of the phenotype in the primate cerebral cortex. Regional specialization in the neuronal phenotype may influence patterns of connectivity and the computational abilities of the circuits they compose. The comparative study of pyramidal cells in homologous cortical areas is beginning to yield data on the evolution and development of such specialized circuitry in the primate cerebral cortex. Recently, we have focused our efforts on sensory-motor cortex. Based on our intracellular injection methodology, we have demonstrated a progressive increase in the size of, the branching structure in, and the spine density of the basal dendritic trees of pyramidal cells through somatosensory areas 3b, 1, 2, 5, and 7 in the macaque and vervet monkeys. In addition, we have shown that pyramidal cells in premotor area 6 are larger, more branched, and more spinous than those in the primary motor cortex (MI or area 4) in the macaque monkey, vervet monkey, and baboon. Here we expand the basis for comparison by studying the basal dendritic trees of layer III pyramidal cells in these same sensory-motor areas in the chacma baboon. The baboon was selected because it has a larger cerebral cortex than either the macaque or vervet monkeys; motor cortex has expanded disproportionately in these three species; and motor cortex in the baboon reportedly has differentiated to include a new cortical area not present in either the macaque or vervet monkeys. We found, as in monkeys, a progressive increase in the morphological complexity of pyramidal cells through areas 3b, 5, and 7, as well as from area 4 to area 6, suggesting that areal specialization in microcircuitry was likely to be present in a common ancestor of primates. In addition, we found subtle differences in the extent of the interareal differences in pyramidal cell structure between homologous cortical areas in the three species. (c) 2005 Wiley-Liss, Inc.

Altered cell surface expression of human MC1R variant receptor alleles associated with red hair and skin cancer risk

The human melanocortin-1 receptor gene (MC1R) encodes a G-protein coupled receptor that is primarily expressed on melanocytes, where it plays a key role in pigmentation regulation. Variant alleles are associated with red hair colour and fair skin, known as the RHC phenotype, as well as skin cancer risk. The R151C, R160W and D294H alleles, designated 'R', are strongly associated with the RHC phenotype and have been proposed to result in loss of function receptors due to impaired G-protein coupling. We recently provided evidence that the R151C and R160W variants can efficiently couple to G-proteins in response to alpha-melanocyte stimulating hormone. The possibility that altered cellular localization of the R151C and R160W variant receptors could underlie their association with RHC was therefore considered. Using immunofluorescence and ligand binding studies, we found that melanocytic cells exogenously or endogenously expressing MC1R show strong surface localization of the wild-type and D294H alleles but markedly reduced cell surface expression of the R151C and R160W receptors. In additional exogenous expression studies, the R variant D84E and the rare I155T variant, also demonstrated a significant reduction in plasma membrane receptor numbers. The V60L, V92M and R163Q weakly associated RHC alleles, designated 'r', were expressed with normal or intermediate cell surface receptor levels. These results indicate that reduced receptor coupling activity may not be the only contributing factor to the genetic association between the MC1R variants and the RHC phenotype, with MC1R polymorphisms now linked to a change in receptor localization.